Process for preparing lincomycin

ABSTRACT

Microbiological process for preparing the antibiotic lincomycin at high incubation temperatures using the micro-organism Streptomyces espinosus and biotypes thereof.

States Patent [1 1 Reusser et al.

[ Sept. 3, 1974 PROCESS FOR PREPARING LINCOMYCIN Inventors: Fritz Reusser; Alexander D.

Argoudelis, both of Portage, Mich.

The Upjohn Company, Kalamazoo, Mich.

Filed: Apr. 23, 1973 Appl. No.: 353,379

Assignee:

US. Cl 195/80 R Int. Cl C12d 9/00 Field of Search 195/80 R References Cited UNITED STATES PATENTS 10/1972 Argoudelis et al 195/80 R Primary Examiner-A. Louis Monacell Assistant Examiner-Robert J. Warden Attorney, Agent, or FirmRoman Saliwanchik 57 ABSTRACT- 8 Claims, N0 Drawings BACKGROUND OF THE INVENTION As disclosed in US. Pat. No. 3,086,912, the antibiotic lincolnens in (lincomycin) can be produced by the micro-organism S. lincolnensis var. lincolnensis, NRRL 2936, at an incubation temperature of l840 C., and preferably at a temperature of 26-30 C. US. Pat. No. 3,697,380 discloses the fermentation production of lincomycin using the micro-organism S. espinosus Dietz, sp. n., NRRL 3890. Again the incubation temperature disclosed for the production of lincomycin is l840 C.

In conducting the above fermentations, it is necessary to use a large amount of cooling water in most fermentation equipment to maintain the desired fermentation temperature. Further, the maintenance of a temperature within the range of l8-40 C., though essential for antibiotic production-as disclosed above, is conducive to the development and proliferation of contaminating microorganisms in the fermentation vessel.

BRIEF SUMMARY OF THE INVENTION TABLE A comycin at a temperature of about 45 C. are Streptomyces pseudogriseolus chemovar linmyceticus, NRRL 3985, and Streptomyces variabilis chemovar liniabilis, NRRL 5618. Thus, the capability of S. espinosus and its biotypes as disclosed herein to produce lincomycin at an incubation'temperature of about 44-48 C. is totally unexpected.

A distinct advantage in using these microorganisms to prepare lincomycin, in addition to the higher fermentation yields of lincomycin shown in Table A, is the need for less fermentor cooling capacity. The need for less cooling capacity is especially significant in high temperature climates and in areas having limited water supplied since water is the generally used means for cooling and maintaining fermentation temperatures.

DETAILED DESCRIPTION OF THE INVENTION 1. The Microorganism The novel actinomycetes used according to this invention for the production of lincomycin are Streptomyces espinosus, NRRL 3890, and three biotypes thereof. One of the strain characteristics of these microorganisms is the production of lincomycin at an incubation temperature range of about 44-48C. A subculture of these living micro-organisms can be obtained upon request from the permanent collection of the Northern Regional Research Laboratories, Agricultural Research Services, US. Department of Agriculture, Peoria, lll., U.S.A. In addition to the above NRRL number, the accession numbers of the S. espinosus biotypes are NRRL 5729,'NRR L 5730, and NRRL 5731.

I Production of Lincomycin by Thermoduric S. espinosus Strains Lincomycin Lincom cin Biotype Temp. Day pH ;Lg/ml* Temp. Day pH pg/m" 22,149a 28C, 1 7.7' S. espino- 28C 1 7.8 2 Y 2 8.7 v 7 sus Y 2 8.8 NRRL S73] 3 9.0 9 NRRL 3890 3 8.9 8

4 9.0 23 (type culture) 4 9.0 17 5 9.0 38- 5 9.1 23

2 9.2 24 2 I 8.7 7 3 9.0 60 3 8.8 59 4 8.8 60 4 8.5 95 5 8.8 110 5 8.3 147 21,987a 28C 1 8.1 2 22,061a 28C 1 7.9 Trace 2 8.6 6 2 8.7 NRRL 5729 3 9.0 2 NRRL 5730 3 9.0 5 4 9.0 8 4 9.0 14 5 9.1 13 5 9.0 19

45C 1 8.3 C 1 8.8 Trace Standard bioassay for lincomycin (S. [urea assay organism, diluent buffer 0.1 M PO pH 7.0, ATCC NOTE: All the ferrnenta'tio'ns used identical seed and fermentation media. The media and other seed and fermentation conditions are shown in Example I, Infra.

The results shown in Table A are unexpected in view The taxonomy of Streptomyces espinosus NRRL 3890 of prior knowledgeof the production of lincomycin by S. espinosus. Also, our tests have shown that S. lincolnensis var. lincolnensis, NRRL 2936, does not produce I lincomycin when incubated at atemperature of about 45 C. Other lincomycin-producing Streptomycetes which produce lincomycin at a normal incubation tempe'rature range, i.e., 1840 C., but do not produce linis disclosed in US. Pat. No. 3,697,380. This microorganism, and the biotypes were studied and characterized by Alma Dietz of the Upjohn Research Laboratories.

DESCRIPTION OF THE MICROORGANISMS Streptomyces espinosus biotpye 21987a, NRRL 5729.

- 3 l Streptomyces espinosus biotype 22061a, NRRL 5730. Streptomyces espiriosus biotype 22149a, NRRL 5731.

1. Color characteristics Aerial growth gray-green. Melanin-negative. Appearance on Ektachrome [Dietz, A. 1954. Ektachrome transparencies as aids in-actinomycete classification. Ann. NY. Acad. Sci. 60:152-154.] is given in Table 1.

Reference color characteristics are given in Tables 2 a and 3. The cultures may be placed in the Green (GN) color series of Tr'esner and Backus [Tresner, H. D., and E. J. Backus. 1063. System of color wheels for streptomycete taxonomy. Applied MicrobioL'l 12335-3381 2. Microscopic characteristics Sporophores short, straight to'flexuous', to open spiral to spiral (RF, RA, S) in the sense of Pridham et al. [Pridham, T. G., C. W. Hesseltine, and R. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains'in morphological sections. Applied Microbiol. 6:52-79]. Spores mostly spherical with many. showing a distinct linkage. Spore surface thorny to spiny in the senseof Dietz and Mathews [Dietz, A., and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Applied Microbiol. 21:527-533]. Some spines show a transition to hairy. Spines are profuse and show markings when observed on spores treated by the carbon replica method of Dietz and Mathews [Dietz, A., and J; Mathews. 1962. Taxonomy by carbon replication. I. An examination of Streptomyces hygroscopicus. Applied Microbiol. 10:258-263].

3. Cultural and biochemical characteristics See Table 4.

Carbon utilization Growth of the cultures on carbon compounds was determined in the synthetic medium of Pridham and Gottlieb [Pridham, T. G., and D. Gottlieb. I948.

(vegetative) in 24 hours and heavy (good sporulation) in 72 hours. The agar media used were Bennetts, Czapeks sucrose, maltose-tryptone. and

Hickey-Tresner (modified).

Source Soils'from southeast Texas.

Type culture Streptomyces espinosus Dietz sp. n., NRRL 3890.

Type variety I Streptomyces espinosus var. espinosus DietzNRRL DISCUSSION M short Sporophores bearing round spiny spores, and

thermoduric growth. These cultures are not considered thermophiles because they grow at temperatures of for infra-subspecific forms based upon differences in physiological or biochemical characteristics.)) of the International Code of Nomenclature of Bacteria [International Code of Nomenclature of Bacteria. 1966. Edited by the Editorial Board of the Judicial Commission of the International Committee on Nomenclature of Bacteria. Intern. J. System. Bacteriol. l.6:459-490]. The isolates are designated Streptomyces espinosus biotype 2l987a, Streptomyces espinosus biotype 2206la, and Streptomyces espinosus biotype 221490.

TABLE 1 Appearance of Streptomyces espinosus Cultures on Ektachrome Agar medium NRRL 3890 NRRL 5729 NRRL 5730 NRRL 5731 Bennetts S Gray-green Graygreen White to gray- Gray-green 7 green R Pale yellow-tan Pale yellow-tan Pale yellow Pale yellow-tan Czapeks S Gray-green Gray-green Gray-green Gray-green sucrose RPale gray Pale gray Pale gray Pale gray Maltose- S Gray-green Gray-green Gray-green Gray-green tryptone R Yellow-tan to Yellow-tan to Yellow Yellow-tan olive olive Peptone-iron S White White White White R Yellow Yellow Yellow Yellow-tan 0.l% Tyrosine S Colorless Colorless Colorless Gray-green R Red Red Red Red Casein starch S Gray-green Gray-green Gray-green Gray-green R Pale gray-green Pale gray-green Pale gray-green "S surface; R reverse Pale gray-green TABLE III 5 Cgntinugd 3rd ed., 1948 Color Code for Table II Color Harmony Manual NBS Circular 553,

Color Color Chip Color name Chip Color name lli Light olive drab l IOgm Grayish olive llca Cream 89gm Pale yellow lxec Biscuit, eeru, oatmeal, sand 90gm Grayish yellow 93m Yellowish gray Wage Dusty yellow 102g Moderate greenish yellow Y l05gm Grayish greenish yellow lVage Light olive gray l09gm Light grayish olive lxie Light olive l96gm Light olive l kig Olive gray 2ca Light ivory eggshell 89gm Pale yellow Zea Light wheat, light maize 86gm Light yellow 20:: Biscuit, eeru, oatmeal, sand 90gm Grayish yellow 2fb Bamboo, buff, straw, wheat 87g Moderate yellow 89m Pale yellow Zfe Covert gray 94g Light olive brown l22gm Light olive gray 2ge Bamboo, ehamois 90gm Grayish yellow 2ge Covert tan, griege 94m Light olive brown l09gm Light grayish olive Zic Honey gold, light gold 87gm Moderate yellow 2ie Light mustard tan 9lgm Dark grayish yellow 94g Light olive brown 106g Light olive 2ig Slate tan 1 lOg Grayish olive l l2m Light olive gray 2ih Dark covert gray 1 12m Light olive gray 1 [3g Olive gray 2le Mustard, old gold 88gm Dark yellow 94g Light olive brown 3ba Pearl, shell tint 3ih Beige gray, mouse 1 13g Olive gray 265m Medium gray 24%fe Light mistletoe gray l22gm Grayish yellow green 24%ih Mistletoe gray 122m Grayish yellow green 127g Grayish olive green "Jacobson, E., W. C. Granville, and C. E. Foss. I948. Color harmony manual, 3rd ed. Container Corporation of America, Chicago, Illinois. *Kelly, K. L., and D. B. Judd. l955.The lSCC-NBS method of designating colors and a dictionary of color names. US. Dept. Comm. Cire. 553;

TABLE IV Cultural and'Bioehemical Characteristics of Streptomyces espinosus Cultures Medium NRRL NRRL NRRL A ar Peptone-iron S Gray-white to Gray-white to Trace gray-green- Gray-green-white gray-green gray-green white R Yellow Yellow Yellow Yellow ,0 Melanin negative Melanin negative Melanin negative Melanin negative Calcium malate S Fair to good gray- Trace gray-green Trace gray-green Trace gray-green green R Pale gray Pale gray Pale gray Pale gray O- Malate not solu- Malate not solu- Malate not solu- Malate not solubilized bilized bilized bilized Glucose- S Gray-white to Gray-white to Fair gray-white Fair gray-white asparagine gray-yellow trace green R Light yellow to Cream Cream Cream cream Skim milk S White on edge to White on edge to White on edge White on edge light gray-green gray-green-pink yellow R Deep yellow to Yellow-tan Yellow-tan Yellow-tan yellow-tan Yellow to yellow- Yellow-tan pig- Yellow-tan pig- Yellow-tan pigtan pigment ment ment ment Casein solubilized Casein solubilized Casein solubilized Casein solubilunder growth to under growth to under growth ized under completely completely growth Tyrosine Good to heavy Good to heavy Good gray-green Good gray-green gray-green gray-green R Red-tan tored- Red-tan to red- Red-tan Red-tan brown brown Red-tan to red- Red-tan to red- Red-tan pigment Red-tan pigment brown pigment brown pigment Tyrosine solu- Tyrosine solu- Tyrosine solu- Tyrosine solubilized bilized bilized bilized Xanthine 5 Good gray-green Good gray-green Good gray-green Good gray-green to good only on periphery R Pale yellow to Pale yellow-green Cream Cream CI'CLIITI 1 l0 cream TABLE IV Continued 4 Cultural and Biochemical Characteristics of Streplomyces espinorus Cultures Medium NRRL NRRL NRRL NRRL 0 Xanthine not Xanthine not Xanthine not Xanthinc not solubilized solubilized solubilized solubilized Nutriehnt 5 Good gray-green Good gray-green Good gray-green Good gray-green stare R Cream-olive Cream-olive Cream-olive Cream-olive 0 Starch hydro- Starch hydro- Starch hydro- Starch hydrolyzed lyzed lyzed lyzed Yeast extract- 5 Good to heavy Good gray-green Good gray-green Good gray-green malt extract gray-green R Pale yellow-tan Cream-yellow-tan Cream-yellow-tan Cream-yellow-tan to cream-yellowtan Bennett's S Cream to heavy Gray-green-white Trace gray-cream Cream gray-green to heavy graygreen R Yellow to olive Cream to dive Yellow Yellow Czapek's S Gray-green Gray-green Gray-green Gray-green sucrose R Gray-green Gray-green Gray-green Gray-green Maltose- S Heavy gray-green Good to heavy Gray-green-white Pale gray-green tryptone gray-green R .Olive to orange- Olive to cream Yellow Yellow-olive yellow Hickey-Tresner S Heavy gray-green Heavy gray-green Gray-green-white Gray-green (modified) 1 R Olive-orange Cream Deep yellow-tan Yellow-olive Peptone-yeast S Cream to pale Pale pink to Cream to pale Cream with trace extract-iron pink gray-green pink gray-green to (lSP-6) pale pink v O Melanin negative Melanin negative Melanin negative Melanin negative Tyrosine S Gray-green-white Gray-green-white Gray-green Gray-green (lSP-7) to gray-green to gray-green R- Pale yellow-green Pale yellow-green Gray-cream Gray-cream to tan to gray-cream O Melanin-negative Melanin-negative Melanin-negative Melanin-negative Gelatin v Plain 0 Colorless vegeta- Colorless vegeta- Gray-white aerial Gray-white aerial tive growth to tive growth to growth; liquegrowth; liquegray-white gray-white faction comfaction comaerial growth aerial growth; pletc plete liquefaction A liquefaction k to complete to complete Nutrient 0 White to gray- White to gray- Gray white aerial Trace gray-white white aerial white aerial growth; liqueaerial growth;

growth; liquegrowth; liquefaction complete liquefaction faction A: to faction $6 to complete complete complete Broth Synthetic 0 White to pink- Trace white to Trace white aerial Trace white aernitrate cream aerial pink-cream growth on surface ial growth on growth on suraerial growth pellicle; growth surface pelface pellicle; on surface pelthroughout medium; licle; trace growth throughliclc; growth nitrate not regrowth throughout medium; nithroughout duced to nitrite out medium; nitrate not remedium, nitrate trate not re duced to nitrite not reduced to duced to nitrite nitrite Nutrient O Gray-green-white Gray-green-white Gray-grcen-white Trace white aernitratc aerial growth aerial growth aerial growth ial growth on on surface pelon surface pelon surface pellisurface pellilicle; flocculicle;-floccucle; flocculent cle; compact lent bottom lent bottom bottom growth; to flocculent growth; nitrate growth; nitrate nitrate not rebottom growth; test: neither not reduced to duced to nitrite nitrate not renitrate nor nitrite duced to ninitrate present trite or nitrate not .reduced to nitrite Litmus milk 0 Cream to green Pink-tan to gray- Tan aerial growth Green-white aeraerial growth on .yellow to tan surface ring; litmus reduced; peptonization; 'pH 7 green aerial growth on surface ring; litmus reduced; peptonization; pH 7.3 v

aS surface, R reverse, P pigment. 0 other characteristics on surface ring; litmus reduced; peptonization; pH 7.3

ial growth on surface ring; partial reduction; partial peptonization; pH

TABLE v NRRL RRL

CONTROL 7 D-Xylose L-Arabinose Rhamnose D-Fructose D-Galactosc D-Glucose D-Mannose Maltose Sucrose Lactose Cellobiose Dextrin l5. Soluble starch Glycerol Ducitol D-Mannitol,

19. D-Sorbitol I lnositol Salicin Phenol Crcsol Na Formate Na Oxalate Na Tartrate Salicylatc Na Acetate Na Citrate Na Succinate 'Pridhum, T. G., and D. Gottlieb. l948. Thc utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56:107l 14.

Good utilization Poor utilization Doubtful utilizatio No growth Results from different studies TABLE Vl Utilization of Carbon Compounds by Slrepmmyces espinvsus Cultures in thc Synthetic Medium of Shirling and Gottlieb TABLE yL- Continued Utilization of Carbon Compounds by Streptomyces espinosus Cultures In the Synthetic Medium of Shirling and Gottlicb' NRRL NRRL NRRL NRRL 3890 5729 5730 573l Raffinosc Cellulose 1,-H- i,'

. Shirling, E. B.. and D. Gottlieb. i966. Methods for churactcrimtinn of SIn-pmmyces species. Int. J. Syst. Bacteriol. l6:3l3:340,

-H- Strong utilization Positive utilization Utilization doubtful Utilization negative Results from different studies I Lincomycin is produced by the novel microorganisms of the subject invention when said microorganisms are grown in an aqueous nutrient medium under sub merged aerobic conditions. It is to be understood also that for the preparation of limited amounts surface cultures and bottles can be employed. The organisms are grown in 'a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and

- a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include com steep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest ofcasein, distillerss solids, animal peptone liquors, fishmeal, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like,'usually need not be added to the fermentation media since tap water and unpurified ingredients are used as media components.

Production of lincomycin by the process of the invention can be effected, advantageously, at a temperature of about 44-48 C., and preferably at about 45 C. Ordinarily, optimum production of lincomycin is obtained in about 2 to 10 days. The medium normally remains basic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.

- When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganisms for inoculation to avoid a pronounced lag in the production of lincomycin and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of lincomycin, as long as it is such that a good growth of the microorganisms is obtained.

The lincomycin produced by the subject process can be recovered by the procedure disclosed in U.S. Pat. No. 3,086,912. v

In preferred recovery process, lincomycin is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means,

such as by filtration and centrifugation. Lincomycin is then recovered from the filtered or centrifuged broth by passing said broth over a resin which comprises a non-ionic macro porous'copolymer of styrene crosslinked with divinylbenzene. Resins of this type are disclosed in US. Pat. No. 3,515,717. Exemplary'of this type of resin is Amberlite XAD-2. Lincomycin is eluted from the resin with a solvent system consisting of methanolwater (95:5. v/v). Bioactive eluate fractions are determined by a standard microbiological disc plate assay using the microorganism Sarcina lutea. Biologically active fractions are combined, concentrated to an aqueous solution which is then freeze dried. The freeze dried material is then triturated with methylene chloride. The methylene chloride extract is concentrated to dryness and the residue triturated with acetone. The filtrate is mixed with ether to give a precipitate which is separated. The remaining filtrate is mixed with methanolic hydrogen chloride (1 N) to precipitate colorless lincomycin hydrochloride. This precipitate is isolated by filtration and crystallized from water-acetone to give crystalline lincomycin hydrochloride.

It is to be understood that the process of the subject invention is not limited to the particular microorganisms fully described by the cultural characteristics dis-' closed herein. It is intended that this invention also include other strains or mutants of the said microorganisms which can be produced by procedures well known in the art, for example, by subjecting the novel microorganisms to x-ray or ultraviolet radiation, nitrogen mustard, phage exposure, and thelike.

Hereinafter is described a non-limiting example of the process of the present invention. All percentages are by weight and all solvent portion mixtures are by volume unless otherwise noted.

Part A. FERMENTATION A soil slant of Streptomyces espinosus, NRRL 3890, is used to inoculate a 500-ml. Erlenmeyer flask con taining 100-ml. of sterile seed medium consisting of the following ingredients:

Glucose monohydrate 25 g./liter Pharmamedia* 25 g./liter Tap water q.s. Balance Presterilization pH 7.2 *Pharmamcdia is an industrial grade of cottonseed flour produced by Traders Oil Mill Company, Fort Worth, Texas.

Kaysoy 35 gm./liter Skim milk gmJliter Czapek Dox broth" 10 gm./liter CaCO:, 3 gm./liter Ucon LB-625*"'"' '20 ml./liter Presterilizati'on pH 7.2 Finely milled fat extracted soybean meal supplied by Archer Daniels Midland Company, 733 Marquette Avenue, Minneapolis, Minnesota. "Supplied by Dilco Laboratories, Detroit, Michigan. *"A polyalkylene glycol defoaming fluid supplied by Union Carbide Corp., Chemicals Division, I042l W. 7 Mile Road, Detroit, Michigan 4822i.

Each flask is inoculated with 5 ml. of seed inoculum per 100 ml. of fermentation medium. Some of the flasks are incubated at 28C. and other at 45 Con a rotary shaker operating at 250 rpm and an eccentricity of 2.5 inches.

The process described above for S. espinosus, NRRL 3890, is repeated for the biotypes, described herein. The results of these fennentations are as shown in Table A, Supra.

Part B. RECOVERY Whole fermentation broth (approximately 4 liters) obtained as described above, is filtered using diatomaceous earth as a filter aid. The filter cake is washed with 1 liter of water and the aqueous wash is combined with the filtrate. The resulting solution is kept as clear beer. The filter cake is triturated twice with 700 ml. of methanol each time. Methanolic extract is kept as MEOH extract. The clear beer is passed over a column containing 250 ml. of Amberlite XAD-Z at a flow rate of 25 ml. per minute. The spent beer is kept as one fraction (spent). The column is then washed with 500 ml. of water. The aqueous wash is kept asone fraction (wash). The column is then eluted with 95 percent aqueous methanol. Fractions of ml. are collected. Results (testing against sensitive S. Jutea) follow:

Zone (mm! Clear Beer 38 Spent 19 Wash 20 Fraction No.

2 l6 4 l4 5 14.5 6 36 7 49 8 51 9 52 10 51 12 47 14 43 16 40 I9 37 20 34 35 29 30 .40 27 26 24 28 24 19 ll 11.5 traces traces 0 Fractions 6-60 are combined. The solution is concentrated to dryness to give 3.5 gms. of a lincomycin preparation assaying 310 mcg. of lincomycin/mg. This material is then triturated with methylene chloride. The methylene chloride extract is concentrated to dryness and the residue triturated with acetone. The filtrate is mixed with ether to give a precipitate which is separated. The remaining filtrateis mixed with methanolic hydrogen chloride (1 N) to precipitate colorless lincoperature range of about 4448 C. until substantial antibiotic activity is imparted to said medium by the production of lincomycin.

-2. A novel process for preparing the antibiotic lincomycin which comprises cultivating Streptomyces espinosus, biotype 2 1987a, having the identifying characteristic of NRRL 5729, and mutants thereof, in an aqueous nutrient medium under aerobic conditions at an incubation temperature range of about 4448 C. until substantial antibiotic activity is imparted to said medium by the production of lincomycin.

3. A novel process for preparing the antibiotic lincomycin which comprises cultivating Streptomyces espinosus, biotype 22061a, having the identifying characteristic of NRRL 5730, and mutants thereof, in an aqueous nutrient medium under aerobic conditions at an incubation temperature range of about 4448 C. until substantial antibiotic activity is imparted to said medium by the production of lincomycin.

4. A novel process for preparing the antibiotic lincomycin which comprises cultivating Streptomyces espinosus, biotype 22l49a, having the identifying characteristic of NRRL 5731 and mutants thereof, in an aqueous nutrient medium under aerobic conditions at an incubation temperature range of about 4448 C. until substantial antibiotic activity is imparted to said medium by the production of lincomycin.

5. A process according to claim 1, wherein the fermentation incubation temperature is about 45 c.

6. A process according to claim 2, wherein the fermentation incubation temperature is about 45 C.

7. A process according to claim 3, wherein the fermentation incubation temperature is about 45 C.

8. A process according to claim 4, wherein the fermentation incubation temperature is about 45 C. 

2. A novel process for preparing the antibiotic lincomycin which comprises cultivating Streptomyces espinosus, biotype 21987a, having the identifying characteristic of NRRL 5729, and mutants thereof, in an aqueous nutrient medium under aerobic conditions at an incubation temperature range of about 44*-48* C. until substantial antibiotic activity is imparted to said medium by the production of lincomycin.
 3. A novel process for preparing the antibiotic lincomycin which comprises cultivating Streptomyces espinosus, biotype 22061a, having the identifying characteristic of NRRL 5730, and mutants thereof, in an aqueous nutrient medium under aerobic conditions at an incubation temperature range of about 44*-48* C. until substantial antibiotic activity is imparted to said medium by the production of lincomycin.
 4. A novel process for preparing the antibiotic lincomycin which comprises cultivating Streptomyces espinosus, biotype 22149a, having the identifying characteristic of NRRL 5731 and mutants thereof, in an aqueous nutrient medium under aerobic conditions at an incubation temperature range of about 44*-48* C. until substantial antibiotic activity is imparteD to said medium by the production of lincomycin.
 5. A process according to claim 1, wherein the fermentation incubation temperature is about 45* c.
 6. A process according to claim 2, wherein the fermentation incubation temperature is about 45* C.
 7. A process according to claim 3, wherein the fermentation incubation temperature is about 45* C.
 8. A process according to claim 4, wherein the fermentation incubation temperature is about 45* C. 